1. Technical Field
The present invention relates to a method for inducing the differentiation of hematopoietic stem cells into megakaryocytes and platelets, a gene controlling the differentiation, and uses thereof.
2. Description of the Related Art
Platelets, playing a crucial role in hemostasis or blood coagulation, measure 2 to 3 μm in diameter, with a mean blood concentration of 300,000 to 500,000 cells/mm2. They are sticky and viscous, and the morphology thereof varies depending on conditions. Platelets adhere and coagulate on damaged tissue and at the same time, release intracellular components which induce a series of coagulation reactions.
Thrombocytopoiesis is the process in which megakaryocytic progenitor cells derived from multipotent stem cells are transformed into megakaryocytes via megakaryoblasts and platelets are then produced from megakaryocytes. The studies of the regulation of megakaryocytopoiesis and thrombocytopoiesis have been carried out by Mazur (Exp. Hematol., 15:248, 1987) and Hoffman (Blood, 74:1196-1212, 1989). For example, bone marrow pluripotent stem cells are differentiated into megakaryocytes, erythrocytes, and myelocytes. Megaloblasts are among the megakaryocytic lineage cells detectable in the early stages of development. These cells have basophilic cytoplasms, reticular chromatin, and one morphologically irregular nucleus containing several nucleoli, and range in diameter from 20 to 30 μm. Within a short time, megakaryocytes have up to 32 nuclei (polyploidy) while the cytoplasm remains largely immature. With the advance of maturation, the nuclei undergo further lobulation and concentration while the cytoplasm increases in volume and is further acidophilic and granulated. In the most mature megakaryocytic lineage cells, platelets are observed to be released from cell verges. Generally, less than 10% of megakaryocytes are in an erythroblastic stage, while more than 50% undergo maturation. Typically, megakaryocytes are morphologically classified into early-stage progenitor megakaryocytes, mid-stage promegakaryocytes or basophilic megakaryocytes, and late-stage mature megakaryocytes (acidophilic, granulate and responsible for platelet biogenesis). Mature megakaryocytes shed cytoplasmic filaments into sinusoidal lumens wherein they are fragmented into individual platelets (Williams et al., Hematology, 1972).
Thrombocytopenia means a reduction in platelet count, caused by the destruction of megakaryocyte colony-forming units, which are the progenitor cells of megakaryocytes present in the bone marrow. Recently, there are several different forms of thrombocytopenia; hereditary thrombocytopenia, idiopathic thrombocytopenic purpura, and aplastic anemia. A clinically more important form of thrombocytopenia is secondary thrombocytopenia which is induced by general irradiation with X-rays, or by the administration of drugs which prevent hematopoiesis. In many cases, secondary thrombocytopenia is caused by chemotherapy, radiotherapy, bone marrow transplantation, or the like applied to cancer patients which results in inadequate formation of bone marrow megakaryocytes. Secondary thrombocytopenia is a dangerous disease which impedes the recovery of the patient and sometimes causes death by bleeding.
A therapy for thrombocytopenia which is currently most frequently used involves platelet transfusion in order to keep the platelet count at a value of more than 20,000/μl. However, it has problems that the donors of blood needed for platelet transfusion are lack, and the infection such as AIDS virus or hepatitis virus derived from blood and immune response according to foreign platelet transfusion occurs.
Therefore, there is a need for the development of substances capable of directly promoting thrombocytopoiesis, which may be directly administered to patients or to the pluripotent hematopoietic stem cells, leading to their differentiation into thrombocytes and an increase in the count.
In contrast, thrombocythemia is a chronic disorder associated with increased or abnormal production of blood platelets. Since platelets are involved in blood clotting, their abnormal production can result in the inappropriate formation of blood clots or in bleeding, with the consequence of an increased risk of gastrointestinal bleeding, heart attack and stroke. Examples of diseases associated with thrombocythemia include essential thrombocythemia (ET), chronic myelogenous (CML), polycythemia vera (PV), agnogenic mteloid metaplasia (AMM), and sickle cell anemia (SCA). To treat thrombocythemia, studies have been made on the compounds inhibiting the platelet production.
Stem cell possesses two properties of multipotency and self renewal, and is found in both embryonic and adult cells. Since one stem cell is able to differentiate into a specialized cell or organ, much interest has focused on organ transplantation or cell therapy using stem cells.
Such platelet-related diseases are caused by increased or decreased production of blood platelets through the specific mechanisms. Therefore, if the genes involved in differentiation of stem cells into megakaryocytes and platelets are used as a target gene for the treatment of thrombocytopenia or thrombocythemia, it is expected that platelet differentiation can be effectively controlled.
Accordingly, intensive and thorough research into thrombocytopoiesis, conducted by the present inventors, resulted in the finding that the compound of Formula 1 of the present invention is highly effective in inducing the differentiation of CD34 positive hematopoietic stem cells into megakaryocytes and thrombocytes. In addition, the present inventors have identified novel genes that are involved in the differentiation of hematopoietic stem cells into megakaryocytes and platelets. They found that the genes can substantially control the differentiation of hematopoietic stem cells into megakaryocytes and platelets, and thus the gene can be used as a target gene for the treatment of thrombocythemia and thrombocytopenia, and as a megakaryocyte/platelet differentiation marker, thereby completing the present invention.